Low Concentrations of Flavonoid - Rich Fraction of Shallot Extract Induce Delayed - Type Hypersensitivity and TH1 Cytokine IFNγ Expression in BALB/c Mice.

Flavonoids are potentially immunomodulatory factors and it may be inferred that these phytochemicals contribute to immunomodulatory properties of the Allium family. In the present study, we investigated the potential mechanism underlying the immunomodulatory effect of shallot and its ethyl acetate (EA) fraction as flavonoid-rich sources. Ex vivo, effects of a hydroalcoholic extract of shallot, its fractions and quercetin on lymphocyte viability were evaluated. The proliferative effects of the fractions were examined using naive mouse lymphocytes to determine the fraction with highest impact/ activity. In addition, in a mouse model, both delayed- type hypersensitivity (DTH) responses and production of a key cytokine (interferon [IFN]-ᵧ) were evaluated. Both the shallot extract and its fractions inhibited lymphocytes cell growth and survival in a concentration- dependent manner. The findings also showed that the extract and especially the ethyl acetate (EA) fraction could induce lymphocyte proliferation. The evaluation of the extract and its EA fraction on DTH responses indicated that both caused a significant increase in DTH response. Furthermore, they triggered significant increases in IFNγ and decreases in interleukin (IL)-4 production by splenic mononuclear cells. Because of the significant immunomodulatory activity displayed in these studies, it is plausible that shallot could have a potential use as an immunomodulatory agent in clinical settings.

involves the interplay of signals among T-cell receptors (TCR), the cytokines interferon (IFN)-γ and interleukin (IL)-12, and the transcription factors T-bet, STAT1 and STAT4 (5). On the other hand, the basis of T H 2 cell differentiation involves interplay of signals among the TCR, IL-4, and transcription factors GATA-3 and STAT6 (6).
Recent studies have shown that T H 1 cell-derived cytokines like IFNγ and IL-12 promote cellular immunity, while T H 2 cell-derived agents like IL-4 and IL-5 induce humoral immunity (7)(8). In The shallot (Allium ascalonicum) is a major component of many Asian diets and is widely believed to be beneficial for health. Like garlic and other members of this family, shallot contains biologically-active components including organosulfur compounds, polyphenols and selenium (14).  (15)(16)(17). Shallots contain considerable amounts of flavonoids that can increase the proliferative activity of lymphocytes in vitro (18). Quercetin is found in high concentrations in shallot and can exert significant immunomodulatory effects, in part, by modulating the production of T H 1 and T H 2 cell-derived cytokines (19).
Previously, we indicated that a flavonoid-rich fraction of a hydroalcoholic extract of shallot could abolish angiogenesis in vitro and in vivo (20).
Furthermore, we reported the results of studies that delineated the in vitro anti-cancer and in vivo antiinflammatory activities of shallot extract (21).
Building upon those earlier findings, the studies here sought to examine the effects of a hydroalcoholic extract of shallot and its flavonoidrich fraction, on induction of delayed typehypersensitivity (DTH) responses and production / release of a key T H 1 cytokine (i.e., IFNγ) in BALB/c mice.  footpad) were monitored after 24 and 48 hr using vernier calipers (27).

Cytokine assay
To evaluate any effects of the shallot extract or the EA fraction on cytokine production by The sensitivity of both the IFNγ and IL-4 kits was 2 pg/ml.

Statistical analysis
All data were presented as mean ± SE.
Statistical analyses were performed for each endpoint using a Student's t-test and a one-way analysis of variance (ANOVA). P-values < 0.05 were considered statistically significant.

Toxicity effect of shallot extract on mouse
The results indicated that a total of five  and 300 ng/ ml ( Figure 2B). The other fractions, as well as the quercetin, had proliferative effects that mirrored the trends noted in the cytotoxicity outcomes (data not shown).

Effect of shallot extract and its EA fraction on DTH responses
To     Figure 4F).

Discussion
The results of several population-based studies have indicated that intake of Allium vegetables is inversely associated with the risk of cardiovascular, diabetes and infectious diseases as well as certain types of cancer (28). Additionally, several studies triggered intense research in the past two decades that aimed not only to identify the putative phytochemicals responsible for the aforementioned health benefits of Allium vegetables but also to elucidate the mechanisms of action (29).
Shallot, as a major component of many Asian diets, is widely believed to be beneficial to health.
Furthermore, it contains flavones and polyphenolic derivatives with anti-cancer and anti-angiogenic properties (30)(31). In previous studies, we showed that a hydroalcoholic extract of shallot and its fractions (especially an ethyl acetate (EA) fraction) exhibited significant anti-angiogenic effects in vitro, ex vivo and also in in vivo models (20,(24)(25).
Additionally, we demonstrated that heat and low pH has had no effect on anti-angiogenic activity of this extract or the EA fraction (20,24). In the present study, it was seen that a hydroalcoholic extract of shallot and its EA fraction each could cause a significant increase in DTH response in mice. In addition, the results showed that each agent could induce significant increases in IFNγ production, and decreases in IL-4 production, by splenic MNC from hosts treated with the test materials. Our laboratories had shown previously that the EA fraction which was rich in flavonoid compounds, imparted significant inhibitory effects on angiogenesis (20). In that study, it was seen that